Skin Dermis
Dermal fibroblasts are cells present in the extracellular matrix within the dermis of the skin. The dermis provides strength and flexibility to the skin and is also a supporting structure for blood vessels, the lymphatic system, nerves, sweat glands and hair follicles. Fibroblasts are the major cell type of the dermis, producing and maintaining the extracellular matrix, which in turn supports other cell types. See Parenteau et al. (2000) “Skin.” Principles of Tissue Engineering. 2nd Ed. Academic Press, San Diego. Fibroblasts secrete various growth factors and cytokines, and produce new extracellular matrix in the granulation tissue. When a wound in the dermis develops, fibroblasts are converted to a contractile myofibroblast phenotype, which initiates wound contraction and epithelization, and leads to complete wound closure.
Etiology of Wounds
Wound healing, or wound repair, is the body's natural process of regenerating dermal and epidermal tissue. When an individual is wounded, a set of events takes place in a predictable fashion to repair the damage. These events overlap in time and must be artificially categorized into separate phases: the inflammatory, proliferative, and maturation phases. See Clark et al. (2000) “Wound repair: Basic Biology to Tissue Engineering.” Principles of Tissue Engineering. 2nd Ed. Academic Press, San Diego.
In the inflammatory phase, bacteria and debris are phagocytosed and removed, and factors are released that cause the migration and division of cells involved in the proliferative phase.
The proliferative phase is characterized by angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction. In angiogenesis, new blood vessels grow from endothelial cells. In fibroplasia and granulation tissue formation, fibroblasts grow and form a new provisional extracellular matrix (ECM) by secreting collagen and fibronectin. In epithelialization, epithelial cells migrate across the wound bed to cover the wound. In contraction, the wound is made smaller by the action of myofibroblasts, which establish a grip on the wound edges and contract themselves using a mechanism similar to that in smooth muscle cells. Unneeded cells undergo apoptosis when the cells' roles are close to complete.
In the maturation phase, collagen is remodeled and realigned along tension lines. Cells that are no longer needed are removed by apoptosis.
Wounds that fail to undergo closure in a normal course of time are termed chronic or non-healing wounds. See Lorenz et al. (2003) Wounds: Biology, Pathology, and Management. Stanford University Medical Center. Lower extremity skin ulcers can result from arterial or venous insufficiency. Factors that affect the repair process in non-healing ulcers are diabetic conditions, ischemia, bacterial infection, and nutrition. For diabetic conditions, the classical risk factors for developing ulcers are peripheral neuropathy, peripheral arterial disease, and susceptibility to infection. See Thuesen A. The University of Montana SPAHS Drug Information Service (2001) 5:1-3.
Treatment of Wounds
The primary goal in the treatment of diabetic foot ulcers is to obtain wound closure. Management of a foot ulcer is largely determined by its severity (grade), vascularity, and the presence of infection. A multidisciplinary approach should be employed because of the multifaceted nature of foot ulcers and the numerous comorbidities that can occur in patients with this type of wound. This approach has demonstrated significant improvements in outcomes, including reduction in the incidence of major amputation.
A mainstay of ulcer therapy is debridement of all necrotic, callus, and fibrous tissue. Unhealthy tissue must be sharply debrided back to bleeding tissue to allow full visualization of the extent of the ulcer and to detect underlying abscesses or sinuses.
Topical applications have been applied for the treatment of diabetic ulcers with some success. Examples include the use of placental extract, which contains various growth factors, and phenyloin for treating non-healing ulcers. See Chauhan et al. Lower Extremity Wounds (2003) 2:40-45. Another topical application, containing recombinant human platelet derived growth factor (PDGF), is Plermin, marketed by Dr. Reddy's Laboratories. Regranex® Becaplermin is the only FDA-approved topical platelet-derived growth factor (PDGF) for chronic diabetic neuropathic ulcers in USA. However, randomized controlled clinical trials showed only a 15% acceleration in the healing of neuropathic diabetic foot ulcers. See Falanga V. Advanced Treatments Chronic Wounds (April 2005)<http://www.worldwidewounds.com/2005/april/Falanga/Advanced-Treatments-Chronic-Wounds.html> February 2006. Although numerous topical medications and gels are promoted for ulcer care, relatively few have proved to be more efficacious than saline wet-to-dry dressings. Topical antiseptics, such as povidone-iodine, are usually considered to be toxic to healing wounds. Topical enzymes have not been proved effective for this purpose and should only be considered as adjuncts to sharp debridement. Soaking ulcers is controversial and should be avoided because the neuropathic patient can easily be scalded by hot water.
Growth factors besides PDGF have not been approved for clinical use and the results of clinical trials have not delivered the expectations generated by preclinical data. See Falanga supra. A possible explanation for this could be that growth factors are required in combination, or a different mode of delivery is required.
Cells are considered “smart materials” and can produce balanced mixtures of different growth factors and cytokines, as well as adapt their responses according to the environment they are in. Cells can themselves help in repairing affected area and damaged tissue. See Falanga, supra. Hence, cell-based applications have the more potential for much better results than the previously mentioned techniques.
With the advent of tissue engineering, there have been promising results shown by different skin substitutes in efficiently treating chronic wounds, which have otherwise been difficult to heal successfully and often lead to amputation of the limb having the ulcer. See Eisenbud et al. Wounds (2004) 16:2-17; Marston et al. Diabetes Care (2003) 26:1701-1705. While skin autografts are successful in effecting wound healing, the autografting procedure is invasive, painful and could lead to a secondary non-healing wound. In the case of chronic wounds, a skin substitute that can act as a temporary biological dressing and trigger tissue regeneration and wound healing by stimulating cells in the patient's own wound bed has the potential to be an effective treatment modality. Cells in the skin substitute may be effective delivery systems for growth factors that would help in stimulating the healing process. Cells that may be used in the skin substitute include dermal fibroblasts and keratinocytes from healthy skin biopsies.
Various skin substitutes have been developed internationally for the treatment of non-healing ulcers. Examples are Apligraf® (Organogenesis Inc.), Dermagraft® (Smith & Nephew Inc.), Oasis® (Healthpoint), and EZ Derm™ (Brennen Medical Inc.). These skin substitutes have shown good clinical results. However the skin substitutes face challenges such as difficult logistics of ordering and using the substitutes due to a cryopreservation requirement, difficulty in maintaining cell viability, poor durability of matrix collagen when exposed to the enzyme-rich wound bed (thereby causing cells to wash away and lose effect), and the thickness of the matrix preventing sufficient diffusion of growth factors from the embedded cells. Another pertinent problem is the cost of these skin substitutes. These challenges have lead to the requirement for improved and innovative solutions. The inventors of the present invention have been successful in providing an improved and alternate solution to the present skin substitutes by providing a temporary biological dressing or wound cover that causes wound healing by stimulating the patient's own tissue to regenerate.
In the emerging field of tissue engineering, there is a requirement for developing tissue equivalents for both in vivo and in vitro uses. In some tissue equivalents that have been developed, there is significant contraction their original size, which limits such equivalents to specific applications in which the contracted construct can be useful. See Clark et al. J. Clin. Invest. (1989) 84:1036-1040; Montesano et al. Proc. Natl. Acad. Sci. (1988) 85:4894-4897. Contracted tissue shrinks inwards immediate after release from a culture surface, while contractile macromass tissue constructs contract to a relatively smaller degree.
The tissue-like organization and constructs previously developed by the present inventors using the novel method of macromass culture (Indian Patent No. 195953 and U.S. patent application Ser. No. 10/686,822, filed Oct. 16, 2003) is an example of a tissue equivalent belonging to this contracting class. The constructs spontaneously reduce in size over a period of time. Another example is a multilayered sheet of keratinocytes, which contracts when detached without support from the culture vessel. See Green et al. Proc. Natl. Acad. Sci. (1979) 76:5665-5668.
Cellular sheets have been cultivated over different supporting layers, such as non-porous sheets. See Khor et al. J. Mater. Sci. Mater. Med. (2003) 14(2):113-20; Imaizumi et al. Tissue Eng. (2004) 10(5-6):657-64. However, non-porous supports limit the supply and diffusion of nutrients and gases. On the other hand, one problem of culturing cells using porous matrices is that a percentage of total cells seeded onto the sponge in the form of cell suspension leak out from the bottom of the sponge onto the base of the culture vessel. This problem has been recognized in earlier work in the field of cell culture methods. See Yang et al. J. Biomed. Mater. Res. (2002) 62(3):438-446. This amounts to loss of cells when seeding, which is an especially critical problem for tissue engineering applications wherein cell sources can be rather limited. Loss of cells would also result in difficulty in getting reproducible constructs from equally seeded sponges, since variable numbers of cells would remain on the sponges if varying numbers of cells are lost while seeding. Methods such as anhydrous ammonia plasma treatment and ethanol treatment have been used for preventing cell loss.
In U.S. Pat. No. 5,273,900, an epidermal cellular sheet was made on one side of a porous collagen dermal substrate, which was prepared by making a non-porous collagen-laminating layer on one side of the porous dermal component to be able to form the epidermal sheet. The laminating layer does not and is not intended to enter into the porous dermal component, and it remains an integral part of the final product.
In another earlier invention involving methods for long-term culture of hematopoietic progenitor cells, the pores of a matrix are filled with a “gelatinous” substance. See Pykett et al., U.S. Pat. Nos. 6,645,489 and 7,192,769. However the gelatinous substance of this invention holds the cells within the pores of the matrix, and there is no sheet formation of the cells on one side of the matrix.
The present invention relates to an advances over the prior art that, for example, allow a tissue substitute to remain viable during transportation without any specific need for cryopreservation. In one embodiment, the present invention presents compositions and methods that addresses the needs of transporting a viable tissue substitute to a recipient's location.